Method of collecting markers of bone metabolism from sweat

ABSTRACT

The invention disclosed herein relates to a method of monitoring bone metabolism comprising continuously collecting a sweat sample from a subject and assaying the sweat sample to determine the concentration of a marker of bone metabolism in the sample, wherein said marker is not pyridinoline or deoxypyridinoline.

This is a continuation-in-part of application Ser. No. 08/263,137 filedon Jun. 21, 1994 U.S. Pat. No. 5,589,346.

TECHNICAL FIELD

The present invention relates to a method for detecting markers of bonemetabolism. More particularly, the present invention relates to methodfor collecting and analyzing sweat for the presence of markers of bonemetabolism.

DESCRIPTION OF RELATED ART

Current methods used to monitor the presence, progress of treatment, ordisease state for metabolic bone diseases require the measurement ofmarkers of bone metabolism found in blood or urine samples. Examples ofthese methods are shown in U.S. Pat. No. 5,283,197 to Robins, U.S. Pat.No. 4,973,666 to Eyre, and U.S. Pat. No. 5,140,103 also to Eyre. Themost commonly measured of these markers include calcium, hydroxyproline,alkaline phosphatase, procollagen Type I and its cleavage products,osteocalcin, and bone collagen peptides that include crosslinked aminoacids. The crosslinked amino acids include pyridinoline, hydroxy lysylpyridinoline, lysyl pyridinoline, N-telopeptide, and the peptides thatcontain the former molecules.

These molecules are specific collagen breakdown products known to beproduced following bone resorption. Thus, the measurement of thesecrosslinked amino acids (markers of bone metabolism) can provide anindication of metabolic bone disease, and can be of use in monitoringthe progress of medical treatment intended to reduce the loss of bonedensity found in various disease states.

As with many other molecules of biological interest, the production ofcrosslinked amino acids varies over time in a diurnal cycle (Schemer etal., 1992, Easel et al., 1992, Easel et al, 1992) and can also vary inconcentration from day-to-day (Hertz et al. 1994). Normal biologicalvariations in the concentration of these collagen breakdown products inhealthy individuals can nearly equal the levels of these molecules thatare obtained in individuals with diagnosed disease states typified byhigh levels of metabolic bone loss over extended periods of time. Suchdisease states include diseases such as osteoporosis (Betake et al.,1992, Deltas, 1991), hyperparathyroidism (Harvet et al., 1991, Robins etal., 1991), Paget's disease (Bettica et al., 1992, Robins et al., 1991),rheumatoid arthritis (Seibel et al., 1989), multiple myeloma (Elomaa etal., 1992), tumor-associated hypercalcemia (Body et al., 1992) andosteoarthritis (Elomaa et al., 1992, Delmas et al., 1991). Theabove-cited U.S. patents to Robins and Eyre describe methods foranalyzing urine and blood samples in order to assay for levels ofindicators of bone loss. However, the collection of a single blood orurine sample is representative of only a single point in time,therefore, any variation in the levels of markers of bone metabolism maynot be discovered due to the natural diurnal and day-to-day variationsin concentration inherent with these markers. Additionally, these priorart methods typically require the services of a technician, such assomeone to draw a blood sample or these prior methods may require thesubject go to a laboratory, a hospital or a doctor's office in order tosubmit a sample for analysis. This can be problematic where a subjectmust submit a series of successive samples for analysis in order toobtain enough test results to allow for a meaningful diagnosis. That is,since the results derived from the prior art methods are representativeof only a single point in time; the subject must submit multiple bloodor urine samples in order reduce the possibility that he/she willreceive a false positive or false negative test result. This problem isnot only one of inconvenience, but also represents a significant cost tothe subject and the potential for misdiagnosis.

In order to overcome the problems associated with the prior art methodof analysis, it is necessary that a modality of collection be introducedwhich eliminates the biological variations due to diurnal and day-to-dayvariations by providing a means of collection which is continuous anduninterrupted thereby allowing for the collection of a more meaningfulsample which has the benefit of being integrated over time to reduce theeffects of biological variations in bone loss marker production.Additionally, the method should also increase the diagnostic power ofthe test by reducing the incidence of false positive and false negativetest results.

The present invention not only eliminates some of the burden andinconvenience associated with the prior art methods, it yields the addedbenefits of improved accuracy and performance by eliminating certainlong-standing problems such as biological variation, thereby making themeasurement of the bone loss marker more meaningful and greatlyincreases its significance in diagnosis and treatment of bone resorptiondisease states. The Applicant has found that markers of bone metabolism(collagen breakdown products) are present in sweat in detectable levelswhich are indicative of physiological bone loss. Additionally,applicants have found that by collecting a sample of sweat over a periodof time, quantifiable results can be obtained which provide for a moreaccurate and representative assessment of bone resorption.

The subject invention describes the use of a skin patch for thecollection of perspiration or sweat which collects a sample for analysisin which the diurnal and day-to-day variations of crosslinked aminoacids is minimized. Perspiration or sweat collected over a period ofdays accumulates in a sample that minimizes the sources of biologicalvariation through integration over time, making the measurement of thebone loss marker more meaningful value and thereby increasing itssignificance in the diagnosis and treatment of disease states. The useof the skin patch also provides a means for specimen collection that canbe performed at clinics, in a physician's office, or at home, thus, itis suitable for monitoring the efficacy of therapeutic regimens formetabolic bone disease and can be used in screening for the onset or theprogression of bone diseases.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a method ofmonitoring markers of bone metabolism by continuously collecting a bodyfluid sample containing markers of bone metabolism therein and analyzingthe components of the body fluids for the markers of bone metabolism.

FIGURES IN THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 is a graph showing the extraction recovery of pyridinoline spikedonto a skin patch where pyridinoline was placed on each patch (n=2) atconcentrations that approximate 20, 50, and 80% binding in theimmunoassay;

FIG. 2 is a bar graph detailing the effect of sunlight on pyridinolinerecovery, pyridinoline was added to duplicate patches at 100 (shadedbars) or 500 fmol/mL (open bars), the data are calculated from theexpected recoveries;

FIG. 3 is a graph showing the effect of sample filtration on measurementof pyridinoline in sweat samples, skin patches worn by volunteers for 1to 7 days were extracted, extracts were spin filtered (abscissa) or notfiltered (ordinate), and pyridinoline was assayed, data from fourseparate experiments were combined; n=22 samples, regression line:y=-0.014+1.02x; r=0.98;

FIG. 4 is a bar graph detailing the recovery of pyridinoline in sweatfrom different parts of the body, patches were worn simultaneously onthe trunk (shaded bars) and extremities for three to five days on eachindividual and each measurement was made in duplicate;

FIG. 5 is a graph detailing the relationship of secretion ofpyridinoline as a function of age, patches were worn by 25 individuals,female: circles, male: boxes;

FIG. 6 is a graph showing the relationship of the concentration ofpyridinoline as a function of time, pyridinoline collected in skinpatches worn for varying lengths of time, patches were worn on the back,extracted and analyzed for pyridinoline;

FIG. 7 is a graph showing the relationship of the concentration ofcreatinine collected in sweat as a function of time, skin patches wereworn concurrently for 1 to 7 days by six subjects and were thenextracted and creatinine was measured in each extract;

FIG. 8 is a graph showing the relationship of the concentration of ureacollected in sweat as a function of time, skin patches were wornconcurrently for 1 to 7 days by six subjects and patches were extractedand urea was measured in each extract;

FIG. 9 is a graph showing the relationship of the concentration ofpotassium collected in sweat as a function of time, skin patches wereworn concurrently for 1 to 7 days by six subjects and were thenextracted and potassium was measured in each extract by use of an ionselective electrode;

FIG. 10 is a graph illustrating a sensitive deoxypyridinolineimmunoassay in which sweat content of the bone loss marker wasquantified wherein ▪ indicates the location on a standard curve of whenfour samples of liquid sweat were assayed;

FIGS. 11A-B illustrates HPLC tracings confirming the presence ofdeoxypyridinoline in sweat wherein (A) shows a sweat sample after beingre-injected for a second HPLC separation after known amounts of standardpyridinoline and deoxypyridinoline were added (spiked) to the sample and(B) shows the same sweat sample as in (A) illustrating the presence oftwo peaks identified as pyridinoline and deoxypyridinoline based ontheir co-migration with pyridinoline and deoxypyridinoline HPLCstandards; and

FIG. 12 is a graph illustrating a standard curve in a specificenzyme-linked immunoassay for for N-telopeptide wherein ▪ represent thelocation on a standard curve of three concentrated (lyophilized) sweatsamples.

DETAILED DESCRIPTION OF THE INVENTION AND ADVANTAGES

The present invention provides a method for monitoring markers of bonemetabolism in a sample of bodily fluid continuously collected over aperiod of time. The subject invention provides a method of continuouslycollecting a bodily fluid, such as sweat, over a period of time in orderto collect a sweat sample containing markers indicative of diseasesinvolving bone loss or resorption or which indicate normal levels ofbone metabolism (i.e., normal growth and death of bone). The collectedsweat sample, then, can be analyzed to determine the presence of thesemarkers of bone metabolism and thereby ascertain possible disease statesor conditions.

The continuous collection of a bodily fluid is defined as theuninterrupted collection of a bodily fluid over a given period of time.A sample of a bodily fluid is collected over a period of time withoutany gaps or breaks in the collection of the sample. This is unlike atypical blood or urine sample which is only representative of the pointin time in which the sample was taken. Continuous and uninterruptedcollection of a bodily fluid allows for the analysis of a sample whichis representative of a period of time and, therefore, diurnal and othervariations over time can be reduced, eliminated, or controlled.

The continuous and uninterrupted collection of a one week long sweatsample for analysis poses a novel and, somewhat less cumbersome methodthan collecting a one week long total urine sample. The continuous anduninterrupted collection of sweat can be done without any subjectparticipation and, therefore, subject compliance can be greatlyenhanced. In other words, all the subject must do is either apply acollection device or have a collection device applied at the beginningof the sampling period and wear the collection device for the prescribedcollection period. After application of the device, the subject isrequired to do nothing else except function normally. Unlike collectingtotal urine samples or successive blood sampling, the subject need onlyvisit his or her clinician at the beginning of the collection period forapplication of the device and at the end of the collection period forremoval and analysis of the sample collected in the device.Alternatively, the subject can apply and/or remove the devicethemselves. Therefore, this minimal amount of subject participationgreatly increases the subject compliance with the sample collection andanalysis thereof.

Perspiration is defined as the functional secretion of sweat due to thesecretory activity of sweat glands. Sweat is the liquid secreted by thesweat glands and primarily contains water and sodium chloride. However,sweat can also contain other compounds or analytes present in trace, butdetectable, amounts such as urea, albumin and markers of bonemetabolism.

Generally, in order to collect a sweat sample, a sweat collection deviceis applied to a subject to be tested. The sweat collection device can beany device suitable for the collection of sweat or perspiration which isalso designed such that the aqueous component (i.e. water) is allowed toevaporate from the collection device leaving behind only non-volatilematter attached to or trapped within an absorbent pad disposed withinthe device. That is, in order to more precisely maintain normal sweat orperspiration rates, the collection device must allow the aqueouscomponent of the sweat to evaporate normally. Any collection devicewhich alters the normal evaporation of sweat or gas exchange of the skinwith its surroundings may induce or lead to the production of inaccuratedata due to abnormal or induced physiological response of the skin tothe artificial environment created by the application of the collectiondevice.

Typical sweat collection devices include an adsorbent pad such as acotton gauze, synthetic pad, other permeable materials, a capillarytube, or skin patch devices, such as those described in U.S. Pat. Nos.4,957,108, 5,076,273, and 5,203,327 all to Schoendorfer et al. and allassigned Sudor Partners, Inc., all of which are incorporated herein byreference. The sweat collection devices disclosed in these patents areall trans-dermal sweat collection devices which allow for the collectionof one or more analytes in a bodily fluid expressed through the skin tobe collected in a patch and concentrated by active and passive drivingoff a substantial portion of the water fraction under the influence ofbody heat (active) and air evaporation (passive). Said another way,these sweat collection devices allow for the recovery of the analytespresent in the sweat while allowing for the normal evaporation of fluidsand gas exchange of the skin with the surrounding atmosphere.

Each of the devices described in the Schoendorfer et al. referencesrefers to a trans-dermal sweat collection patch which contains anabsorbent pad for collection of analytes present in the collected sweatsamples which is affixed to the subject by means of an adhesive such asadhesive tape or other means known to those skilled in the art. Analytespresent in the sweat samples are deposited on the absorbent pad and theaqueous and volatile contents of the sweat are eliminated by evaporationand normal gaseous exchange with the environment, respectively.

The sweat collection patches are applied over the prepared area of skinand are worn by the subject for a prescribed collection time period.

After the patch has been worn for a sufficient period of time, the patchis removed from the subject and is prepared for analysis. The absorbentpad is separated from an adhesive backing and is prepared for analysisby placing the absorbent pad into a vacuum desiccator to ensure uniformdehydration. The analytes, i.e. markers of bone metabolism, are thenextracted from the absorbent pad in a suitable extraction buffer inorder to remove the analytes containing the markers of bone metabolismfrom the pad area by methods described below.

Markers of bone metabolism include any products produced or given offduring normal or abnormal growth and/or death of bone. These markers ofbone metabolism can be used to determine normal, healthy conditions aswell disease states. The markers of bone metabolism may be any of agroup including crosslinked amino acids, such as pyridinoline, hydroxylysyl pyridinoline, lysyl pyridinoline, substituted pyridinolines,N-telopeptide, procollagen Type I and its cleavage products, andosteocalcin. Some preferred markers for use in the method of the presentinvention include pyridinoline and N-telopeptide since they arecompounds known to be indicative of bone resorption and are present inthe sweat in amounts which are detectable and representative ofresorptive bone diseases. However, the other markers described below arealso indicative of bone metabolism and when viewed in light of theexamples set forth below, cover a number of the known markers of bonemetabolism.

Pyridinoline, deoxypyridinoline, and N-telopeptide are markers whichindicate bone-loss. These markers are catabolic products derived fromthe enzymatic digestion of bone-type collagen by a specialized bonedegrading cell, i.e., osteoclasts. The peptides produced by osteoclastsare further degraded to free cross-links (pyridinoline anddeoxypyridinoline) in blood, liver, and predominately in the kidney. Thepresence of these molecules in blood, urine, and sweat indicates thatbone loss or remodeling is occurring.

After the markers of bone metabolism, i.e., pyridinoline, have beenextracted from the absorbent pad, the extract containing the markers ofbone metabolism is analyzed by enzyme immuno-assay (EIA), HPLC, or byany other method well known in the art which is capable ofquantitatively and/or qualitatively detecting the presence of desiredmarkers, in order to quantitatively determine the concentration of thebone loss marker and qualitatively determine the presence of markers ofbone metabolism.

In order to more accurately determine the concentration of the bone lossmarker present in a given sweat sample, the volume of sweat isnormalized by quantitatively analyzing the concentration of a referencemarker in the sweat collected concurrently with the sweat sample fromthe subject. The reference marker may be any analyte secretedtransdermally at a relatively constant and known rate. Such a referenceanalyte can typically include creatinine, urea, potassium or any otheranalyte secreted at a constant rate. By quantitatively measuring theconcentration of a specific reference marker, it is possible todetermine the volume of sweat which has been transdermally secreted overthe given collection period. Knowing the approximate volume of sweatsecreted during this time period provides an accurate estimate of thevolume of sweat collected thereby allowing an accurate calculation ofthe concentration of the bone loss marker present in the sample.Therefore, a quantitative value for the concentration of a bone lossmarker, integrated over a given time period, is obtained which is freeof diurnal and day-to-day variations. This method provides for thecontinuous and uninterrupted collection of a sample over a period oftime which eliminates the uncertainty and variability associated with a"point in time" sampling method such as a blood or urine analysis.

EXAMPLES Materials and Methods

REAGENTS

From Sigma Chemical Co. (St. Louis, Mo.) sodium phosphate, thimerosal,Triton X-100, Tween 20 and assay kits for the determination ofcreatinine and urea. The osmolarity of samples was measured by use of avapor pressure osmometer (Wescor model 5500, Logan Utah).

SWEAT SAMPLE COLLECTION

The area of the skin to which skin patches were applied was wiped for-30 seconds with a sterile alcohol prep pad (Professional Disposables,Inc., Orangeburg, N.Y.), and the skin was allowed to dry completely (˜2min.) before patch application. Care was taken not to touch theabsorbent pad of the skin patch at any time. Patches were applied bystretching the skin slightly to eliminate wrinkles. Each patch wasplaced over the prepared area and was smoothed from the center towardthe periphery. The time of skin patch application was recorded, andpatches typically were removed at 24 hour intervals, ±1/2 hour. Padswere separated from their adhesive backing and were stored in plasticbags at 4° C.

EXTRACTION

Skin patches (Sudormed, Inc., Santa Ana, Calif.) were dried in a vacuumdesiccator overnight to assure uniform hydration. The patches wereplaced in 3 mL syringes (Becton Dickinson Company, Rutherford, N.J.)along with 1.0 mL (1.0-2.5 ml) of the extraction buffer (EB; 10 mM NaPO₄pH 7.4, 0.02% thimerosal, 0.1% Triton X-100). The syringes were agitatedon a rotary platform shaker at 180 rpm for 3 hours at room temperaturein the dark, and the extract was expelled for analysis. Patch extractswere filtered by centrifugation through 0.1 μm Whatman (Clifton, N.J.)spin filters at 2000×g for 10 min. Extracts were stored at 4° C. in thedark.

RECOVERY FROM SKIN PATCHES

100 μL aliquots of solutions containing pyridinoline and creatinine wereplaced onto duplicate skin patches. The patches were dried in a vacuumdesiccator overnight and then were extracted with 1.0 mL of EB. Sampleswere analyzed for pyridinoline by EIA and for creatinine using alkalinepicrate.

ANALYTICAL METHODS

Pyridinoline. Pyridinoline in sweat was determined by modifying anenzyme immunoassay kit designed to measure pyridinoline in serum(Special Edition Collagen Crosslinks™ Kit; Metra Biosystems, Inc.Mountain View, Calif.). Pyridinoline standards and controls from the kitwere diluted 500-fold with extraction buffer. In coated microliterwells, 100 μL (50-200 μL) of standard, control or sample was reactedovernight at 4° C. with 50 μL (25-150 μL) of primary antibody. Afterwashing the wells, 150 μL of second antibody-enzyme conjugate was added.Following one hour incubation at room temperature, the wells were washedand 150 μL of substrate (p-nitrophenol) solution was added. Theabsorbance at 405 nm was measured in a plate reader (Titertek MultiskanPlus, ICN Biomedicals, Costa Mesa, Calif.) after a one hour incubation.

Deoxypyridinoline. Deoxypyridinoline was detected using two independentanalytical approaches. The first was enzyme-linked immunoassay using amonoclonal antibody specific for this bone-loss marker. The manufacturerof the assay kit (Metra Biosystems) has shown the specificity of theantibody. The antibody has an affinity constant for deoxypyridinoline of3×10⁸ and shows cross reactivity with pyridinoline of less than 2%.Likewise, the antibody shows negligible cross-reactivity (<2.5%) withincompletely digested bone collagen peptides containingdeoxypyridinoline. Deoxypyridinoline has been detected in liquid sweatcollected during periods of heat-or exercise-induced sweating. It wasmeasured by immunoassay using a specific monoclonal antibody raisedagainst human deoxypyridinoline isolated from urine (Metra Pyrilinks-D®,Metra Biosystems, Inc.; cf. Gomez et al., 1996).

N-telopeptide. This peptide is a precursor to free cross-links(pyridinoline and deoxypyridinoline) and is derived from bone-collagen.Its concentration in blood and in sweat and urine is thus directlyrelated to the rate of bone resorption. The amount of N-telopeptide wasmeasured in these fluids with a specific monoclonal antibody(Osteomark®, Ostex, Inc.; cf. Rosen et al., 1994). Applicants collectedN-telopeptide in sweat by two different methods. First, in two adultsand in one male adolescent, N-telopeptide was measured in exercise- andheat-induced liquid sweat (first concentrated by lyophilization) whereit ranged between 0.26-0.89 pmoL/mL (=0.04-0.21 pmoL/μmoL K⁺ ; K⁺ wasused as a marker of sweat volume, 4 μmoL of K⁺ being equivalent to 1 mlof liquid sweat). The second method of sweat collection utilized theOsteopatch ™ (Pacific Biometrics, Irvine, Calif.), an absorbent deviceapplied to skin). Extracts of the patches obtained from seven subjectscontained 0.08-0.91 pmoL/μmoL K⁺ of N-telopeptide. Concentrated liquidsweat was used to confirm the presence of N-telopeptide and related bonepeptides by HPLC. In all cases, the amounts of bone loss markers werecorrected back to the unconcentrated condition, i.e., as it existed infreshly collected liquid sweat.

Creatinine. Creatinine was measured using minor variations of a commonclinical method using the Jaffe reaction (alkaline picrate; MetraBiosystems, Mountain View, Calif.). The creatinine standard was diluted10, 100 and 1000-fold to include the low level of the analyte found insweat samples. In microliter wells we placed 50 μL of standards orsample and 100 μL alkaline picrate, and absorbance at 492 nm wasmeasured in the Titertek plate reader.

Urea. The urea nitrogen assay kit (Sigma) was used to measure both ureaand ammonia in sweat samples, based on the hydrolysis of urea intoammonia and carbon dioxide. The total concentration of urea and ammonia(T) was measured by reacting samples with urease, followed by reactionwith hypochlorite and phenol which, in the presence of sodiumnitroprusside, forms the blue chromophore, indophenol. Indophenol wasquantified by absorbance at 600 nm. Ammonia (A) was measured by omittingurease in the same reaction scheme. The amount of urea in a sample priorto urea hydrolysis (U) was calculated by subtracting the ammonia valuefrom the total value found for both ammonia and urea (U=T-A).

Potassium. Potassium in sweat was determined by means of an ion specificelectrode (Cole Parmer Instrument Company, Niles, Ill.). The electrodewas calibrated against dilutions of a 1.0 g/L potassium standard (ColeParmer).

Results

Recovery of pyridinoline spiked onto unworn skin patches. Pyridinolinerecovery in the patch extracts was nearly quantitative when patches wereextracted with EB (FIG. 1). Similar results were obtained using 0.1%Tween 20 instead of Triton X-100 and also when the buffer was omitted.We selected EB as the standard method due to the stabilizing effect ofthe buffer and the lack of interference of Triton X-100 on theimmunoassay (data not shown).

SOURCES OF ASSAY INTERFERENCE

It is possible that artificially low or high levels of pyridinoline orof reference markers was measured due to sample instability,contamination or other assay interference. Significant potential sourcesof error include: a) photolysis of pyridinoline by ultraviolet light; b)the effect of skin enzymes or bacterial action on analyte levels; and c)the effect of shed epidermis on measurements.

Elimination of photolysis. Pyridinoline was spiked onto patches thatwere then exposed to direct sunlight for 21 hours (FIG. 2). Patches thatwere shaded by aluminum foil gave no significant loss of analyte. Mostpyridinoline in uncovered patches was lost, and covering patches withthin cloth did not completely protect the pyridinoline. Thus, for thepurpose of measuring pyridinoline in sweat, the skin patch shouldinclude a layer of material that is opaque to ultraviolet light toprevent photolysis of pyridinoline.

Effect of bacterial action. Compounds that accumulate in the skin patchare exposed to conditions that might favor growth of microorganisms.

Effect of cellular debris. Pyridinoline is found in collagen from avariety of tissues, but it has not been found in skin. While there maybe no pyridinoline in skin, it is possible that cellular debriscollected in the patch will interfere non-specifically in thepyridinoline assay. To test for the presence of such assay interference,skin patches were worn by volunteers, the patches were extracted andaliquots of the extracts were filtered through Whatman nitrocellulosespin filters (0.1 μm) and assayed for pyridinoline. Results of thecomparison of filtered and nonfiltered samples (FIG. 3) suggest thatthere may be no significant assay interference due to protein andcellular debris in the extract; the calculated values average 103% ofthe measured values for nonfiltered samples.

MEASUREMENT OF MARKERS OF BONE METABOLISM IN SWEAT SAMPLES

Pyridinoline levels based on location on the body. Levels ofpyridinoline in sweat collected in skin patches worn on the trunk(abdomen and lower back) and on the extremities (upper arms and legs).In all individuals tested, the mass of pyridinoline was greater inpatches worn on the trunk than those on the extremities (FIG. 4).

Pyridinoline levels based on age. Levels of pyridinoline measured insweat collected in from a variety of individuals, ranging in age from 2to 50 years. In all cases, skin patches were worn on the lower back. Themass of pyridinoline was greater in very young children, and, amongadults, highest among females (FIG. 5).

Pyridinoline levels based patch contact time. Skin patches worn forlonger time accumulate a greater mass of pyridinoline (FIG. 6). In allcases, skin patches were worn on the lower back. The rate ofaccumulation of pyridinoline was similar in many cases.

Because of the relatively low abundance of deoxypyridinoline comparedwith pyridinoline, it was necessary to concentrate liquid sweat bylyophilization (freeze-drying) prior to assay.

In three volunteers, deoxypyridinoline ranged from 0.14 to 0.24 pmoL/mL(picomol=10⁻¹² moL). In one adolescent undergoing rapid bone growth (andtherefore, displaying a high rate of bone remodeling), deoxypyridinolinewas found to be 0.78 pmoL/mL. The amount of deoxypyridinoline wassmaller than the amount of pyridinoline measured in the same samples inaccordance with the greater content of pyridinoline in bone collagen.

The presence of deoxypyridinoline was independently confirmed by HPLC(High Performance Liquid Chromatography) using the standard methodsdeveloped for the detection of deoxypyridinoline in bone digests and inhuman urine (Eyre, 1987). In two concentrated sweat samples,deoxypyridinoline by HPLC was 0.23 and 0.50 pmoL/mL. Within the accuracyof the method, these values confirmed the immunoassayable content ofsweat. In all cases, the amounts of bone loss markers were correctedback to the unconcentrated condition, i.e., as it existed in freshlycollected liquid sweat.

FIG. 10 illustrates the results of a sensitive deoxypyridinolineimmunoassay in which sweat content of the bone loss marker wasquantified. The abscissa is the concentration of validated standards ofdeoxypyridinoline provided by Metra Biosystems. The concentration ofstandards are pmoL/mL (=nM; =10⁻⁹ M) The ordinate is B/B_(o), a measureof the specific displacement of assay antigen from the antibody assample or standard antigen was increased. The square symbols indicatethe location on the standard curve of when four samples of liquid sweatwere assayed. Three of the samples were concentrated by lyophilizationfor before assay. The sample showing the lowest deoxypyridinoline valuewas assayed directly without concentration.

FIG. 11 illustrates two HPLC (High Performance Liquid Chromatography)tracings which confirmed the presence of deoxypyridinoline in sweat byan independent method. HPLC was the reference method for measurement ofbone-loss markers in bone digests, serum and urine. The lowerchromatogram in each Figure represents the optical density of fractionsmeasured by fluorescence (excitation 295 nm; emission 395 nm). The uppertracing is the optical density in the ultraviolet (205 nm). Thepyridinium nucleus of pyridinoline and deoxypyridinoline is fluorescentand, therefore, can be monitored by fluorescence. In this case, the UVrecording was done as other (non-fluorescent) compounds in sweat werebeing sought at.

FIG. 11B shows the presence of two peaks identified as pyridinoline anddeoxypyridinoline based on their co-migration with pyridinoline anddeoxypyridinoline HPLC standards.

FIG. 11A shows the same sample of sweat re-injected for a second HPLCseparation after known amounts of standard pyridinoline anddeoxypyridinoline were added (spiked) to the sample. The specificelevation in peak height and peak area of the two peaks indicated thatthe smaller co-migrating peaks in the lower Figure are bona fidepyridinoline and deoxypyridinoline.

FIG. 12 shows the standard curve in a specific enzyme-linked immunoassayfor N-telopeptide (Osteomark, Ostex, Inc.) As in the deoxypyridinolineassay, this is a competitive assay in which increasing amounts ofantigen in standards of sample lower the signal (B/B_(o)). The squaresymbols indicate the location on the standard curve of threeconcentrated (lyophilized) sweat samples. Also indicated are the extentto which the three samples were concentrated prior to lyophilization.

MEASUREMENT OF REFERENCE MARKERS

Three candidate compounds were selected creatinine, urea and potassium,based on their high concentrations in sweat and relatively constantconcentrations at varying sweat rates. It is possible that none of theseanalytes will suffice as a sweat volume marker, and there are others wemight need to test, but this will be known only after sufficient datahave been collected. In addition to these analytes, we will test theutility of specific gravity of patch extracts as an index of sweatvolume.

Creatinine in sweat. Levels of creatinine in sweat did not increaserapidly with an increase in the length of time a patch was worn (FIG.7).

Urea in sweat. The mass of urea increased at about the same rate for allindividuals tested, leveling off when patches were applied for greaterthan five days (FIG. 8).

Potassium in sweat. As with urea, the levels of potassium in sweat fromseveral individuals increased with the length a patch was worn, and theaccumulation of potassium dropped off when patches were worn for greaterthan five days (FIG. 9).

The experimental data set forth above provides support for the genericterm "markers of bone metabolism" and, therefore, provides sufficientsupport for broad coverage of all markers of bone metabolism detectablein sweat.

The invention has been described in an illustrative manner, and it is tobe understood the terminology used is intended to be in the nature ofdescription rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings.

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We claim:
 1. A method of monitoring bone metabolism comprisingcontinuously collecting a sweat sample from a subject and assaying thesweat sample to determine the concentration of a marker of bonemetabolism in the sample, wherein said marker is not pyridinoline ordeoxypyridinoline.
 2. The method of claim 1 further including the stepof normalizing the volume of the sweat sample collected.
 3. The methodof claim 1 wherein said normalizing step further includes quantitativelyanalyzing the concentration of a reference marker present in the sweatsample, which amount is directly associated with the amount of sweatcollected from the subject.
 4. The method of claim 3 wherein thereference marker is selected from the group consisting of creatinine,urea, potassium, and other suitable markers.
 5. The method of claim 1wherein the marker of bone metabolism is n-telopeptide.
 6. The method ofclaim 1 wherein the marker of bone metabolism is lysyl pyridinoline. 7.The method of claim 1 wherein the marker of bone metabolism issubstituted pyridinoline.
 8. The method of claim 1 wherein the marker ofbone metabolism is hydroxylysyl pyridinoline.
 9. The method of claim 1wherein the marker of bone metabolism is procollagen Type I and itscleavage products.
 10. The method of claim 1 wherein the marker of bonemetabolism is osteocalcin.
 11. The method of claim 1 wherein said stepof collecting sweat further includes applying a sweat collection meansto the subject.
 12. The method of claim 11 wherein the sweat collectionmeans is selected from the group consisting of a skin patch, anabsorbent pad, or a capillary tube.
 13. The method of claim 1 furthercomnrises extracting the marker of bone metabolism from the collectedsweat sample.
 14. The method of claim 13 wherein said assaying stepcomprises qualitatively and quantitatively determining the concentrationof the marker of bone metabolism.
 15. The method of claim 14 wherein theconcentration of the marker of bone metabolism is determined by enzymeimmunoassay techniques.
 16. A method of using a sweat collection deviceby collecting a sweat sample from a subject wherein the sweat samplecontains markers of bone metabolism; andassaying the sweat sample formarkers of bone metabolism.
 17. A method for determining the volume ofsecreted sweat over a given period of time by quantitatively determiningthe concentration of at least one reference marker secreted in sweatcollected over the given time period.
 18. The method of claim 17,wherein said step of quantitatively determining the concentration of atleast one reference marker is further defined as of normalizing thevolume of the sweat sample collected.
 19. The method of claim 18,wherein said normalizing step further includes quantitatively analyzingthe concentration of a reference marker present in the sweat sample,which amount is directly associated with the amount of sweat collectedfrom the subject.
 20. The method of claim 19, wherein the referencemarker is selected from the group consisting of creatinine, urea,potassium, or other suitable markers.
 21. The method of claim 17,wherein said step of collecting a sweat sample includes applying a sweatcollection device to the subject.
 22. The method of claim 21, whereinthe sweat collection device is isolated from the group consisting of askin patch, an absorbent pad, and a capillary tube.